The present invention relates to a method of delivering a benefit agent to a surface. More in particular, it relates to a method whereby a benefit agent is loaded to a first surface and subsequently unloaded and transferred and delivered to a second surface. In a preferred embodiment, it relates to the transfer of benefit agent, loaded on to a garment during the washing process, and subsequent delivery of the benefit agent to another surface.
Conventionally, benefit agents, such as bleach and perfume, are incorporated in detergent compositions, adsorbed onto surfaces, and act on the garments during the washing process. After the washing process, the effects are generally short-lived. In addition, large quantities of benefit have to be present to achieve an effect.
WO-A-98/56885 (Unilever) discloses a bleaching enzyme which is capable of generating a bleaching chemical and having a high binding affinity for stains present on fabrics, as well as an enzymatic bleaching composition comprising said bleaching enzyme, and a process for bleaching stains on fabrics. The binding affinity may be formed by a part of the polypeptide chain of the bleaching enzyme, or the enzyme may comprise an enzyme part which is capable of generating a bleach chemical that is coupled to a reagent having the high binding affinity for stains present on fabrics. In the latter case the reagent may be bispecific, comprising one specificity for stain and one for enzyme. Examples of such bispecific reagents mentioned in the disclosure are antibodies, especially those derived from Camelidae having only a variable region of the heavy chain polypeptide (VHH), peptides, peptidomimics, and other organic molecules. The enzyme which is covalently bound to one functional site of the antibody usually is an oxidase, such as glucose oxidase, galactose oxidase and alcohol oxidase, which is capable of forming hydrogen peroxide or another bleaching agent. Thus, if the multi-specific reagent is an antibody, the enzyme forms an enzyme/antibody conjugate which constitutes one ingredient of a detergent composition. During washing, said enzyme/antibody conjugate of the detergent composition is targeted to stains on the clothes by another functional site of the antibody, while the conjugated enzyme catalyzes the formation of a bleaching agent in the proximity of the stain and the stain will be subjected to bleaching.
WO-A-98/00500 (Unilever) discloses detergent compositions wherein a benefit agent is delivered onto fabric by means of peptide or protein deposition aid having a high affinity for fabric. The benefit agent can be a fabric softening agent, perfume, polymeric lubricant, photosensitive agent, latex, resin, dye fixative agent, encapsulated material, antioxidant, insecticide, soil repelling agent, or a soil release agent. The benefit agent is attached or adsorbed to a peptide or protein deposition aid having a high affinity to fabric. Preferably, the deposition aid is a fusion protein containing the cellulose binding domain of a cellulase enzyme. The compositions are said to effectively deposit the benefit agent onto the fabric during the laundering process.
According to DE-A-196 21 224 (Henkel), the transfer of textile dyes from one garment to another during a washing or rinsing process may be inhibited by adding antibodies against the textile dye to the wash or rinse liquid.
WO-A-98/07820 (PandG) discloses amongst others rinse treatment compositions containing antibodies directed at cellulase and standard softener actives (such as DEQA). WO 99/27368 describes the use of a displaceable moiety able to bind to 2 different surfaces. However, all interactions are specific (to an analyte of interest and a mimitope) and the assay is specifically aimed at measuring an analyte of interest for a Unipath application.
There is a need for extending the length of time that a benefit acts on the garment after the washing process. There is also a need to transfer the benefit agent from the garment onto another surface, for example during wearing or storage of the garment, thereby extending the scope of benefit that can be delivered and achieved.
Surprisingly, it has now been found that antibodies can bind to one surface through non-specific charge interaction and then unload on to a second surface through specific e.g. antigen/antibody interactions. Opportunities for other non-specific interactions include e.g. charge, hydrophobicity/hydrophilicity, trapping due to size constraints.
According to the invention, a benefit agent is first loaded to a surface and subsequently unloaded and transferred and delivered to a second surface. The benefit agent is chosen to impart a benefit onto the surface, which may be a garment, skin, or a ligand thereof. For skin applications, this benefit agent can be bleach, moisturisers, skin softeners (e.g. silicones), emollients, sunscreens, lipids, vitamins, anti-microbial agents, anti-aging benefits, anti-perspirants, skin lightening agents, fabripseuticals, skin-sensory cues (such as menthol, capsasin, silicones) and chemicals. For laundry, the beneft agent can be in the form of a bleaching agent (produced by, for example, bleaching enzymes) that can de-colourise stains, fragrances, colour enhancers, fabric regenerators, softening agents, finishing agents/protective agents, and the like. These will be described in more detail below. The benefit agent may be encapsulated in sensitised particles, bound directly to reagent as a fusion construct, or bound to antibody as a bi-head (WO-A-99/23221). The term antibody includes monoclonal and antibody fragments (scFv, Fab, Fv, VHH, camelised VH).
The benefit agent is loaded onto the first surface by means of a carrying agent. The reagents carrying the benefit agent can bind specifically or non-specifically to surfaces and then bind to a second surface via specific or non-specific interactions. The possible scenarios are depicted in FIG. 1, which shows the binding of the carrying agent/benefit agent through non-specific interaction (A, C) to the first surface and the subsequent unloading of carrying agent/benefit agent through non-specific (C) or specific (A) interactions with a second surface.
Where specific interactions are involved for binding to both surfaces, then displacement to the second surface may result from the reagent having an increased affinity for the second surface over the first. The unloading of the benefit agent may result from pH changes, pressure/abrasion, affinity of the benefit being greater for the second surface than the reagent. Surfaces can be loaded with benefit agent through protein, antibody, peptide, DNA or carbohydrate interactions.
For laundry applications, the primary surface to be loaded with antibody is the fabric. Specifically for laundry applications, the reagent may be loaded all over the garment or be targeted to a specific site, e.g. a site of damage or the underarm region.
As used herein, the term xe2x80x9cmulti-specific binding moleculexe2x80x9d means a molecule which at least can associate onto fabric and also capture benefit agent. Similarly, the term xe2x80x9cbi-specific binding moleculexe2x80x9d as used herein indicates a molecule which can associate onto fabric and capture benefit agent.
In the first step the binding molecule carrying the benefit agent is directly delivered to the fabric, for example a garment, preferably at relatively high concentration, thus enabling the loading of the benefit agent to the fabric in an efficient way.
Examples of the second surface for the subsequent loading include skin, microbes, lipids, steroids, fabric, ligand thereof. For non-laundry applications, the surfaces can be plastic, metal, polystyrene (exemplified in Example 3), hair (whereby the second surface could be a yeast causing dandruff), or a cleaning cloth whereby the second surface is a microbe. Another way of carrying out the invention is to use binding molecules to remove unwanted components from the first surface, e.g. soil or microbes.
In a second step, the carrying agent is contacted with the benefit agent, which may be contained in a dispersion or solution, preferably an aqueous solution, or in a dry environment, e.g. tranfer to skin from garment whilst in wear, thus enabling the benefit agent to bind to the binding molecule through another specificity of said binding molecule.
The multi-specific binding molecule can be any suitable molecule with at least two functionalities, i.e. having a high binding affinity to the fabric to be treated and being able to bind to a benefit agent, thereby not interfering with the pre-determined activity of the benefit agent and possible other activities aimed. In a preferred embodiment, said binding molecule is an antibody, or an antibody fragment, or a derivative thereof. The present invention can be advantageously used in, for example, treating stains on fabrics, preferably by bleaching said stains. In a first step, the binding molecule is applied, preferably on the stain. The benefit agent which is then bound to the binding molecule preferably is an enzyme or enzyme part, more preferably an enzyme or enzyme capable of catalyzing the formation of a bleaching agent under conditions of use. The enzyme or enzyme part is usually contacted to the binding molecule (and the stains) by soaking the pre-treated fabric into a dispersion or solution comprising the enzyme or enzyme part. The dispersion or solution which usually but not necessarily is an aqeous dispersion or solution also comprises ingredients generating the bleaching agent, or such ingredients are added later. Preferably, the enzyme or enzyme part and said other ingredients generating a bleach are contained in a washing composition, and the step of binding the enzyme (or part thereof) to the binding molecule and generating the bleaching agent is performed during the wash. Alternatively, the benefit agent may be added prior to or after washing, for example in the rinse or prior to ironing.
The targeting of the benefit agent according to the invention which in this typical example is a bleaching enzyme, results in a higher concentration of bleaching agent in the proximity of the stains to be treated, before, during or after the wash. Alternatively, less bleaching enzyme is needed as compared to known non-targeting or less efficient targeting methods of treating stains.
Another typical and preferred example of the use of the present invention is to direct a fragrance (such as a perfume) to fabric and to a second surface, e.g. skin, so that it is released over time. A further typical use of the present invention is treating a surface, e.g. fabric where the colour is faded by directing a benefit agent to the area in order to colour that region. Similarly, a damaged area of a fabric can be (pre-)treated to direct a repair of cellulose fibers which are bound by the antibodies to this area. These agents are for example suitably added to the pre-treated fabric after washing, in the rinse.
Other applications, such as using fabric softening agents, polymeric lubricants, photoprotectove agents, latexes, resins, dye fixative agents, encapsulated materials antioxidants, insexticides, soil repelling agents or soil release agents, as well as other agents of choice, and ways and time of adding the agents to the pre-treated fabric are fully within the ordinary skill of a person skilled in the art.
In order to be more fully understood, certain elements of the present invention will be described hereinafter in more detail. Reference is also made to WO 98/56885, referred to above, the content of which is incorporated herewith by reference.
1.0 Binding Molecules
In the first step according to the invention a multi-specific binding molecule is delivered to fabric, said binding molecule having a high affinity to said area through one specificity.
The degree of binding of a compound A to another molecule B can be generally expressed by the chemical equilibrium constant Kd resulting from the following reaction:             [      A      ]        +          [      B      ]        ⇔      [          A      ≡      B        ]  
The chemical equilibrium constant Kd is then given by:       K    d    =                    [        A        ]            xc3x97              [        B        ]                    [              A        ≡        B            ]      
Whether the binding of a molecule to the fabric is specific or not can be judged from the difference between the binding (Kd value) of the molecule to one type of fabric, versus the binding to another type of fabric material. For applications in laundry, said material will be a fabric such as cotton, polyester, cotton/polyester, or wool. However, it will usually be more convenient to measure Kd values and differences in Kd values on other materials such as a polystyrene microtitre plate or a specialised surface in an analytical biosensor. The difference between the two binding constants should be minimally 10, preferably more than 100, and more preferably, more that 1000. Typically, the molecule should bind to the fabric, or the stained material, with a Kd lower than 10xe2x88x924 M, preferably lower than 10xe2x88x926 M and could be 10xe2x88x9210 M or even less. Higher binding affinities (Kd of less than 10xe2x88x925 M) and/or a larger difference between the one type of fabric and another type (or background binding) would increase the deposition of the benefit agent. Also, the weight efficiency of the molecule in the total composition would be increased and smaller amounts of the molecule would be required.
Several classes of binding molecules can be envisaged which deliver the capability of specific binding to fabrics, to which one would like to deliver the benefit agent. In the following we will give a number of examples of such molecules having such capabilities, without pretending to be exhaustive. Reference is also made in this connection to WO-A-98/56885 (Unilever), the disclosure of which is incorporated herein by reference.
1.1 Antibodies
Antibodies are well known examples of compounds which are capable of binding specifically to compounds against which they were raised. Antibodies can be derived from several sources. From mice, monoclonal antibodies can be obtained which possess very high binding affinities. From such antibodies, Fab, Fv or scFv fragments, can be prepared which have retained their binding properties. Such antibodies or fragments can be produced through recombinant DNA technology by microbial fermentation. Well known production hosts for antibodies and their fragments are yeast, moulds or bacteria.
A class of antibodies of particular interest is formed by the Heavy Chain antibodies as found in Camelidae, like the camel or the llama. The binding domains of these antibodies consist of a single polypeptide fragment, namely the variable region of the heavy chain polypeptide (VHH). In contrast, in the classic antibodies (murine, human, etc.), the binding domain consist of two polypeptide chains (the variable regions of the heavy chain (VH) and the light chain (VL)). Procedures to obtain heavy chain immunoglobulins from Camelidae, or (functionalized) fragments thereof, have been described in WO 94/04678 (Casterman and Hamers) and WO 94/25591 (Unilever and Free University of Brussels).
Alternatively, binding domains can be obtained from the VH fragments of classical antibodies by a procedure termed xe2x80x9ccamelizationxe2x80x9d. Hereby the classical VH fragment is transformed, by substitution of a number of amino acids, into a VHH-like fragment, whereby its binding properties are retained. This procedure has been described by Riechmann et al. in a number of publications (J. Mol. Biol. (1996) 259, 957-969; Protein. Eng. (1996) 9, 531-537, Bio/Technology (1995) 13, 475-479). Also VHH fragments can be produced through recombinant DNA technology in a number of microbial hosts (bacterial, yeast, mould), as described in WO-A-94/29457 (Unilever).
Methods for producing fusion proteins that comprise an enzyme and an antibody or that comprise an enzyme and an antibody fragment are already known in the art. One approach is described by Neuberger and Rabbits (EP-A-0 194 276). A method for producing a fusion protein comprising an enzyme and an antibody fragment that was derived from an antibody originating in Camelidae is described in WO-A-94/25591. A method for producing bispecific antibody fragments is described by Holliger et al. (1993) PNAS 90, 6444-6448.
WO-A-99/23221 (Unilever) discloses multivalent and multispecific antigen binding proteins as well as methods for their production, comprising a polypeptide having in series two or more single domain binding units which are preferably variable domains of a heavy chain derived from an immunoglobulin naturally devoid of light chains, in particular those derived from a Camelid immunoglobulin.
An alternative approach to using fusion proteins is to use chemical cross-linking of residues in one protein for covalent attachment to the second protein using conventional coupling chemistries, for example as described in Bioconjugate Techniques, G. T. Hermanson, ed. Academic Press, Inc. San Diego, Calif., USA. Amino acid residues incorporating sulphydryl groups, such as cysteine, may be covalently attached using a bispecific reagent such as succinimidyl-maleimidophenylbutyrate (SMPB), for example. Alternatively, lysine groups located at the protein surface may be coupled to activated carboxyl groups on the second protein by conventional carbodiimide coupling using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
A particularly attractive feature of antibody binding behaviour is their reported ability to bind to a xe2x80x9cfamilyxe2x80x9d of structurally-related molecules. For example, in Gani et al. (J. Steroid Biochem. Molec. Biol. 48, 277-282) an antibody is described that was raised against progesterone but also binds to the structurally-related steroids, pregnanedione, pregnanolone and 6-hydroxy-progesterone. Therefore, using the same approach, antibodies could be isolated that bind to a whole xe2x80x9cfamilyxe2x80x9d of stain chromophores (such as the polyphenols, porphyrins, or caretenoids as described below). A broad action antibody such as this could be used to treat several different stains when coupled to a bleaching enzyme.
1.2 Fusion Proteins Comprising a Cellulose Binding Domain (CBD)
Another class of suitable and preferred binding molecules for the purpose of the present invention are fusion proteins comprising a cellulose binding domain and a domain having a high binding affinity for another ligand. The cellulose binding domain is part of most cellulase enzymes and can be obtained therefrom. CBDs are also obtainable from xylanase and other hemicellulase degrading enzymes. Preferably, the cellulose binding domain is obtainable from a fungal enzyme origin such as Humicola, Trichoderma, Thermonospora, Phanerochaete, and Aspergillus, or from a bacterial origin such as Bacillus, Clostridium, Streptomyces, Cellulomonas and Pseudomonas. Especially preferred is the cellulose binding domain obtainable from Trichoderma reesei. 
In the fusion protein according to the invention, the cellulose binding domain is fused to a second domain having a high binding affinity for another ligand. Preferably, the cellulose binding domain is connected to the domain having a high binding affinity for another ligand by means of a linker consisting of about 0-20, preferably about 2-15, more preferably of 2-5 amino acids.
The second domain having a high binding affinity to another ligand may, for example, be an antibody or an antibody fragment. Especially preferred are heavy chain antibodies such as found in Camelidae.
The CBD antibody fusion binds to the fabric via the CBD region, thereby allowing the antibody domain to bind to corresponding antigens that comprise or form part of the benefit agent.
The fusion protein may comprise more than one cellulose binding domain and an antibody fragment or derivative thereof, or coversely one cellulose binding domain fused to more than one antibody in that the antibody may have the same or different specificities.
1.3 Peptides
Peptides usually have lower binding affinities to the substances of interest than antibodies. Nevertheless, the binding properties of carefully selected or designed peptides can be sufficient to provide the desired selectivity to bind a benefit agent or to be used in an aimed process, for example an oxidation process.
A peptide which is capable of binding selectively to a substance which one would like to oxidise, can for instance be obtained from a protein which is known to bind to that specific substance. An example of such a peptide would be a binding region extracted from an antibody raised against that substance. Other examples are proline-rich peptides that are known to bind to the polyphenols in wine.
Alternatively, peptides which bind to such substances can be obtained by the use of peptide combinatorial libraries. Such a library may contain up to 1010 peptides, from which the peptide with the desired binding properties can be isolated. (R. A. Houghten, Trends in Genetics, Vol 9, no and, 235-239). Several embodiments have been described for this procedure (J. Scott et al., Science (1990) 249, 386-390; Fodor et al., Science (1991) 251, 767-773; K. Lam et al., Nature (1991) 354, 82-84; R. A. Houghten et al., Nature (1991) 354, 84-86).
Suitable peptides can be produced by organic synthesis, using for example the Merrifield procedure (Merrifield (1963) J.Am.Chem.Soc. 85, 2149-2154). Alternatively, the peptides can be produced by recombinant DNA technology in microbial hosts (yeast, moulds, bacteria) (K. N. Faber et al. (1996) Appl. Microbiol. Biotechnol. 45, 72-79).
1.4 Peptidomimics
In order to improve the stability and/or binding properties of a peptide, the molecule can be modified by the incorporation of non-natural amino acids and/or non-natural chemical linkages between the amino acids. Such molecules are called peptidomimics (H. U. Saragovi et al. (1991) Bio/Technology 10, 773-778; S. Chen et al. (1992) Proc.Natl.Acad. Sci. USA 89, 5872-5876). The production of such compounds is restricted to chemical synthesis.
1.5 Other Organic Molecules
The list on proteins and peptides described so far are by no means exhaustive. Other proteins, for example those described in WO-A-00/40968, which is incorporated herein by reference, can also be used.
It can be readily envisaged that other molecular structures which need not be related to proteins, peptides or derivatives thereof, can be found which bind selectively to substances one would like to oxidise with the desired binding properties. For example, certain polymeric RNA molecules which have been shown to bind small synthetic dye molecules (A. Ellington et al. (1990) Nature 346, 818-822). Such binding compounds can be obtained by the combinatorial approach, as described for peptides (L. B. McGown et al. (1995), Analytical Chemistry, 663A-668A).
This approach can also be applied for purely organic compounds which are not polymeric. Combinatorial procedures for synthesis and selection for the desired binding properties have been described for such compounds (Weber et al. (1995) Angew. Chem. Int. Ed. Engl. 34, 2280-2282; G. Lowe (1995), Chemical Society Reviews 24, 309-317; L. A. Thompson et al. (1996) Chem. Rev. 96, 550-600). Once suitable binding compounds have been identified, they can be produced on a larger scale by means of organic synthesis.
2.1 Bleaching Enzymes
As mentioned above, the benefit agent can be bleach, moisturisers, skin softeners (e.g. silicones), emollients, sunscreens, lipids, vitamins, anti-microbial agents, anti-aging benefits, anti-perspirants, skin lightening agents, fabripseuticals, and chemicals. Suitable bleaching enzymes which are useful for the purpose of the present invention are capable of generating a bleaching chemical.
The bleaching chemical may be hydrogen peroxide which is preferably enzymatically generated. The enzyme for generating the bleaching chemical or enzymatic hydrogen peroxide-generating system is generally selected from the various enzymatic hydrogen peroxide-generating systems which are known in the art. For example, one may use an amine oxidase and an amine, an amino acid oxidase and an amino acid, cholesterol oxidase and cholesterol, uric acid oxidase and uric acid, or a xanthine oxidase with xanthine. Alternatively, a combination of a C1-C4 alkanol oxidase and a C1-C4 alkanol is used, and especially preferred is the combination of methanol oxidase and ethanol. The methanol oxidase is preferably isolated from a catalase-negative Hansenula polymorpha strain. (see for example EP-A-244 920 of Unilever). The preferred oxidases are glucose oxidase, galactose oxidase and alcohol oxidase.
A hydrogen peroxide-generating enzyme could be used in combination with activators which generate peracetic acid. Such activators are well-known in the art. Examples include tetraacetylethylenediamine (TAED) and sodium nonanoyl-oxybenzenesulphonate (SNOBS). These and other related compounds are described in fuller detail by Grime and Clauss in Chemistry and Industry (Oct. 15, 1990) 647-653. Alternatively, a transition metal catalyst could be used in combination with a hydrogen peroxide generating enzyme to increase the bleaching power. Examples of manganese catalysts are described by Hage et al. (1994) Nature 369, 637-639.
Alternatively, the bleaching chemical is hypohalite and the enzyme is then a haloperoxidase. Preferred haloperoxidases are chloroperoxidases and the corresponding bleaching chemical is hypochlorite. Especially preferred chloroperoxidases are vanadium chloroperoxidases, for example from Curvularia inaequalis. 
Alternatively, peroxidases or laccases may be used. The bleaching molecule may be derived from an enhancer molecule that has reacted with the enzyme. Examples of laccase/enhancer systems are given in WO-A-95/01426. Examples of peroxidase/enhancer systems are given in WO-A-97/11217.
Suitable examples of bleaches include also photobleaches. Examples of photobleaches are given in EP-A-379 312 (British Petroleum), which discloses a water-insoluble photobleach derived from anionically substituted porphine, and in EP-A-035 470 (Ciba Geigy), which discloses a textile treatment composition comprising a photobleaching component.
2.2 Fragrances
The benefit agent can be a fragrance (perfume), thus through the application of the invention it is able to impart onto the fabric and second surface a fragrance that will remain associated with the surfaces for a longer period of time than conventional methods. Fragrances can be captured by the binding molecule directly, more preferable is the capture of xe2x80x9cpackagesxe2x80x9d or vesicles containing fragrances. The fragrances or perfumes may be encapsulated, e.g. in latex microcapsules.
2.3 Colour Enhancers
The benefit agent can be an agent used to replenish colour on garments. These can be dye molecules or, more preferable, dye molecules incorporated into xe2x80x9cpackagesxe2x80x9d or vesicles enabling larger deposits of colour.
2.4 Fabric Regenerating Agents
The benefit agent can be an agent able to regenerate damaged fabric. For example, enzymes able to synthesize cellulose fibres could be used to build and repair damaged fibres on the garment.
2.5 Others
A host of other agents could be envisaged to impart a benefit to the second surface, e.g. fabric or skin. These will be apparant to those skilled in the art and will depend on the benefit being captured at the fabric surface. Examples of softening agents are clays, cationic surfactants or silicon compounds. Examples of finishing agents/protective agents are polymeric lubricants, soil repelling agents, soil release agents, photo-protective agents (sunscreens), anti-static agents, dye-fixing agents, anti-bacterial agents and anti-fungal agents.
3.1 The Surfaces
For laundry detergent applications, several classes of natural or man-made fabrics can be envisaged, in particular cotton. Such macromolecular compounds have the advantage that they can have a more immunogenic nature, i.e. that it is easier to raise antibodies against them. Furthermore, they are more accessible at the surface of the fabric than for instance coloured substances in stains, which generally have a low molecular weight.
An important embodiment of the invention is to use a binding molecule (as described above) that binds to several different types of fabrics. This would have the advantage of enabling a single benefit agent to be deposited to several different types of fabric.
The skin""s natural substrates could be used to activate the benefit agent once it comes into contact with the skin.